The present invention relates generally to improved methods for the expression of recombinant protein products under the transcriptional control of an inducible promoter, such as the araB promoter, in bacterial host cells that are deficient in one or more of the active transport systems for an inducer. In the case of the araB promoter, the inducer is L-arabinose. The present invention also relates to improved bacterial host cells that are deficient in one or more of the active transport systems for an inducer, such as L-arabinose, and that contain an expression vector encoding a recombinant protein product under the transcriptional control of an inducible promoter, such as the araB promoter.
Although many systems have been described for expression of recombinant proteins, including peptides and polypeptides, in microbial systems, most gene expression systems in gram negative bacteria such as Escherichia coli have relied exclusively on a limited set of bacterial promoters. The most widely used bacterial promoters have included the lactose [lac] (Yanisch-Perron et al., 1985, Gene 33: 103-109), and the tryptophan [trp] (Goeddel et al., 1980, Nature (London) 287: 411-416) promoters, and the hybrid promoters derived from these two [tac and trc] (Brosius, 1984, Gene 27: 161-172; and Amanna and Brosius, 1985, Gene 40: 183-190). Other commonly used bacterial promoters include the phage lambda promoters PL and PR (Elvin et al., 1990, Gene 37: 123-126), the phage T7 promoter (Tabor and Richardson, 1998, Proc. Natl. Acad. Sci. U.S.A. 82: 1074-1078), and the alkaline phosphatase promoter [pho] (Chang et al., 1986, Gene 44: 121-125). Each of these promoters has desirable features. However, the ideal promoter for expression of a wide variety of recombinant proteins would offer certain features not found in these commonly used systems. For example, many recombinant products can be toxic to the expression host. Therefore, it is often preferable for the promoter to tightly regulate gene expression during culture propagation when gene expression is undesirable. In contrast, when gene expression is desired, the promoter must be easily controlled and a high expression level is often preferred. The agent or environmental condition that initiates gene expression should be easy to use and ideally of low cost. In general, a tightly regulated system is most desirable. Features of a promoter and general expression system that are most preferred include tightly repressed gene expression in the absence of inducer and highly derepressed gene expression in the presence of inducer. Also desirable would be a system that will allow the expression level of a recombinant product to be proportional to the amount of inducing agent added into the cell culture.
One bacterial promoter system that has proven to be particularly advantageous for providing tightly repressed gene expression in the absence of the inducer arabinose and highly derepressed gene expression in the presence of the inducer arabinose is the araB promoter of the Enterobacteriaceae family. Of interest is U.S. Pat. No. 5,028,530, which is hereby incorporated by reference, which describes the use of the araB expression system for production of polypeptides, including cecropins, by microbiological techniques. Two features of the ara system have made it particularly well-suited for expression of recombinant products in bacteria such as E. coli. First, it is simple to exploit because the control elements of the araB promoter are conveniently contained within an approximately 300 base pair regulatory region and only a functional coding sequence for the araC gene is additionally needed. Second, regulation of the system has proven to be particularly tight, i.e., the ratio of the amount of product in the induced state (with arabinose) relative to that in the repressed state (without arabinose) from the araB promoter on multicopy expression plasmids is relatively high, most frequently in the range from  greater than 200-75,000 (Better et al., 1999, in Gene Expression Systems: Using Nature for the Art of Expression, Academic Press, New York, pp. 95-107). Additionally, the uninduced level of protein expression from the ara system is very low. This feature is particularly important and useful when protein products, including recombinant peptides and polypeptides, that are toxic to the host are to be expressed.
Bacteria such as E. coli have two known systems for active transport of arabinose into the cell. The first of these systems is an inducible, energy-dependent accumulation process catalyzed by the product of the araE gene. The araE gene product is an approximately 52,000 Da, membrane-associated protein that constitutes the low affinity arabinose transport system (Maiden et al., 1988, Journal of Biol. Chem. 263: 8003-8101). The second L-arabinose transport system has a greater affinity for L-arabinose and is dependent on the activity of the L-arabinose binding protein, AraF. The locus encoding this arabinose binding protein, araF, is part of an operon with araG and araH. The protein products from these three genes, araFGH, make up the high affinity L-arabinose transport system (Horazdovsky and Hogg, 1989, Jour. of Bacter. 171: 3053-3059). Arabinose transport-deficient mutant E. coli strains have been prepared (see, e.g., Maiden et al., supra; Harazdovsky and Hogg, supra).
Of interest are the disclosures of the following references which relate to use of the araB promoter for expression of polypeptides in bacteria.
Johnston et al., 1985, Gene 34: 137-145, described the vector pING1 which contained the ara regulatory region, the complete araC gene and a portion of the araB gene from S. typhimurium. Restriction sites were introduced into the coding region of araB gene so that a gene fusion or a multigene transcription unit could be expressed under arabinose control. This system was used to express homologous (bacterial) proteins which normally are expressed in E. coli under certain circumstances, namely the M13 gene II (Johnson et al., 1985) and gene 8 proteins (Kuhn and Wickner, 1985, J. Biol. Chem. 260: 15907-15918), and a similar vector were used to express the RepE protein from the E. coli F plasmid (Masson et al., 1986, Nucleic Acids Res. 14: 5693-5711). Each of these proteins was produced in the cytoplasm of E. coli cells, and at least some of the expressed protein was soluble and could be detected in an active form either in vivo or in cell extracts.
Better et al., 1988, Science 240: 1041-1043, described an araB expression system derived from pING1 that was subsequently engineered to regulate the expression of heterologous (non-bacterial) recombinant proteins. Heterologous proteins successfully produced with the araB expression system in E. coli include immunoglobulin Fab domains. In this case, the araB expression system was used to direct the production of polypeptides directly linked to hydrophobic signal sequences through the bacterial cytoplasmic membrane where Fab accumulated in the correctly folded, fully active configuration and could be recovered directly from the culture supernatant. Expression of Fab domains under the transcriptional control of the araB promoter was the first demonstration that a heterodimeric, heterologous protein could be produced in E. coli. The initially reported expression level was approximately 1-2 xcexcg/mL, but in subsequent studies the level of protein expression could be increased nearly 1000-fold by growing the bacteria to a high cell density in a fermentor (Better et al., 1990, ICSU Short Rep. 10: 105). In contrast, Clark et al., 1997, Immunotechnology 3: 217-226, found that Fab genes under lac control can inhibit bacterial growth, and also that Fab expression from PBAD was more tightly repressed than that from Plac.
Better et al., 1992, J. Biol. Chem. 267: 16712-16718; Nolan et al., 1993, Gene 134: 223-227; and Bernhard et al., 1994, Bioconjugate Chem. 5: 126-132, showed that the araB expression system could be used successfully for the production of other proteins. Several plant and fungal ribosome-inactivating proteins were expressed under direct control of arabinose in E. coli. One such protein, gelonin, was expressed as a secreted protein in E. coli and accumulated to greater than 1 g/L as a fully active protein in the cell-free culture supernatant. Fusion proteins between antibody domains that can target antigens on human cells and cytotoxic molecules such as gelonin were also expressed under the transcriptional control of arabinose, Better et al., 1995, J. Biol. Chem. 270: 14951-14957. These immunofusion proteins accumulated in the cell-free culture supernatant from arabinose-induced cells at greater than 400 mg/L. Members of a family of immunofusion proteins expressed in E. coli under the transcriptional control of the araB promoter retained both in vitro and in vivo biological activity comparable to that of chemically prepared immunoconjugates made from animal cell-produced whole antibodies and ribosome-inactivating proteins purified directly from plants.
Jacobs et al., 1989, Gene 83: 95-103 and Romeyer et al., 1990, Appl. Environ. Microbiol. 56: 2748-2754, also used plasmids derived from pING1 to encode mammalian proteins that can become localized in the outer cell membrane of bacteria. In one example, a human metallothionein-II gene was linked to the leader and membrane-association fragment of the E. coli lipoprotein Lpp (Jacobs et al., 1989). When induced with arabinose, bacteria carrying this expression vector directed an active metallothionein protein to the outer cell membrane. The recombinant protein was produced at xcx9c75,000-fold over the uninduced level. This system was used to express an active heterologous protein that previously had been somewhat toxic and unstable in E. coli. 
Cagnon et al., 1991, Protein Eng. 4: 843-847, described a series of expression vectors that contained the ara expression system from pING1 in the vector pKK233.2 along with a number of other optional features. In the Cagnon series of expression vectors, the promoter/operator region of araB was followed by a polylinker region for convenient gene cloning. In addition, some vectors contained synthetic signal sequences, an f1 phage origin of replication, and mutated araB promoter sequences. The mutated araB promoter incorporated changes in the xe2x88x9210 region that made the promoter match more closely a consensus E. coli promoter. The promoter mutations resulted in higher level of inducible expression (2-fold) for a marker gene, however, the uninduced expression level increased as well. Several recombinant proteins were expressed from this family of ara expression vectors including the full length Tat protein from the HIV virus (Armengaud et al., 1991, FEBS Lett. 282: 157-160) and the bacterial proteins: xcex2-galactosidase (Cagnon et al., 1991), the Streptoalloteichus hindustanus bleomycin-binding protein (Cagnon et al., 1991), and the cholera toxin subunit B (Slos et al., 1994, Protein Express. Purif. 5: 518-526). The cholera toxin subunit B (CT-B) was linked to the ompA signal sequence and expressed as a secreted protein. CT-B accumulated to approximately 60% of the total periplasmic protein and CT-B was produced at about 1 g/liter at pilot scale. The majority of CT-B was released into the culture medium and could be recovered at greater than 80% efficiency from the cell-free culture medium.
Perez-Perez and Gutierrez, 1995, Gene 158: 141-142, described an ara expression system in pACYC184 that remains compatible with ColE1-derived plasmids in an expression host.
Guzman et al., 1995, J. Bacteriol. 177: 4121-4130, described a series of araB expression vectors that incorporate various selectable markers and multicloning sites. This series of vectors was studied extensively for the expression of native E. coli proteins. Guzman et al. (1995) also presented evidence that the araB system can be used xe2x80x9cto achieve very low levels of uninduced expression, obtain moderately high levels of expression in the presence of inducer, and modulate expression over a wide range of inducer concentrations.xe2x80x9dThe possibility was raised that the extent of arabinose induction can be regulated by the amount of inducer added to the culture.
Others have reported that gene expression under the control of the araB promoter appeared to be directly regulated by the concentration of arabinose introduced into the culture medium (Lutz and Bujard, 1997, Nucleic Acids Research 25: 1203-1210; and Carrier et al., 1998, Biotechnol. Bioengineering 59: 666-672). However, in contrast, Siegele and Hu, 1997, Proc. Natl. Acad. Sci. U.S.A. 94: 8168-8172, demonstrated that gene expression from plasmids containing the araB promoter at subsaturating arabinose concentrations actually represent the population average of mixed populations of induced and uninduced cells. Thus, intermediate expression levels in culture simply reflected the average of the induced and uninduced cells in the population, not direct regulation. Siegele and Hu, supra, constructed plasmids that contained a reporter gene for a mutated green fluorescent protein (gfp), and those plasmids expressed a fast-folding mutant of the Aequorea victoria green fluorescent protein under the transcriptional control of the araB promoter. Microscopic examination of cells grown at low arabinose concentrations showed a mixture of brightly fluorescent and dark cells, suggesting that intermediate expression levels in cultures reflect a population average. Because the inducer arabinose is actively transported into the cell, the amount of induction in any given cell would be expected to vary with the amount of arabinose actively transported into that cell. Thus, the average amount of induction in a population of cells would, in turn, represent the average amount of arabinose actively transported into that population of cells. It is important to note, however, that those cells that do contain high concentrations of arabinose are fully induced. A similar phenomenon has also been reported with lactose transport and the induction of the lac operon (Novick and Weiner, 1957, Proc. Natl. Acad. Sci. USA 43: 553 and Maloney and Rotman, 1973, J. Mol. Biol. 73: 71-91). Thus, these references, including Siegele and Hu, supra, conclude that although araB vectors have the advantages of rapid regulation and low basal level expression compared to plasmids regulated by the lac repressor, the araB promoter is not well-suited to modulate the expression of cloned genes because of variances in arabinose uptake between cells.
The references cited above indicate that the araB expression system is useful for the controlled expression of recombinant proteins in bacterial systems. These references further indicate that the araB system offers advantages not found in other bacterial expression systems. However, a problem still facing the art is to generate directly regulated cell cultures in which the amount of protein expression in the cells is actually proportional to the amount of inducer (e.g., arabinose) present in culture medium. Because induction of recombinant products in bacterial cells can often inhibit subsequent cell growth, it would be desirable to generate cell cultures in which gene expression could be regulated directly and proportionally by the amount of inducer present.
The present invention is directed to improved methods for the expression of recombinant protein products, including peptides and polypeptides, under the transcriptional control of an inducible promoter, such as the araB promoter. According to the invention, bacterial host cells are genetically engineered with deficiencies in active transport system(s) for an inducer of a promoter contain vectors for the expression of recombinant protein products under the transcription control of an inducible promoter. The present invention specifically provides novel genetically engineered bacterial host cells, that are genetically deficient in one or both of the two known L-arabinose transport systems in E. coli and related bacteria of the Enterobacteriaceae family, including species of Salmonella, Pseudomonas, Proteus and Enterobacter, and capable of expressing recombinant protein products. Use of such transport-deficient bacterial host cells, including arabinose transport-deficient bacterial host cells, for the expression of recombinant protein products under the transcriptional control of an inducible promoter such as the araB promoter provides, for increased expression of the recombinant protein product, lower inhibition of host cell growth after induction, and/or direct and synchronous induction of expression in such host cells as compared to that in the transport-proficient cells. Recombinant protein products, including protein products useful as therapeutic, prophylactic and/or diagnostic agents, are thus efficiently and economically produced according to the methods of the present invention.
The present invention provides a method for producing a recombinant protein product under the inducible control of an inducible promoter, such as an araB promoter, comprising: (a) introducing a recombinant expression vector encoding a recombinant protein product under the control of an inducible (e.g., araB) promoter into a bacterial host cell that is genetically deficient in at least one system for active transport of the inducer (e.g., arabinose) into the host cell; and (b) inducing expression of the recombinant protein product with the inducer (e.g., arabinose).
According to the invention, an improved method provides direct regulation, including synchronous induction, of bacterial cell cultures, in which expression of recombinant protein product is proportional to the culture concentration of the inducer, (e.g., arabinose). In genetically engineered host cells according to the invention that contain a vector for protein product expression under the transcription control of the inducible (e.g., araB) promoter and that are deficient in inducer (e.g., arabinose) transport, the intracellular concentration of the inducer (e.g., arabinose) is proportional to the concentration of the inducer (e.g., arabinose) in the culture medium because passive diffusion is the only mechanism for intracellular accumulation of the inducer (e.g., arabinose) across the bacterial membranes. This is direct regulation of expression and leads to synchronous induction of recombinant protein product expression in the host cells. Thus, the methods of the present invention offer advantages not found with expression in transport-proficient bacterial hosts from an inducible promoter, such as the araB promoter, where there is a mixed population of induced and uninduced cells, and where the apparent relationship between the amount of recombinant product and the concentration of inducing agent at subsaturating amounts of the inducer, such as arabinose, reflects the population average of expression in both induced and uninduced cells.
The present invention also provides a bacterial host cell that is deficient in one or more of the active transport systems for an inducer, such as L-arabinose, and that contains an expression vector encoding a recombinant protein product under the transcriptional control of an inducible promoter, such as the araB promoter. Such cells are useful for the production of a variety of protein products. According to the invention, recombinant protein products may be therapeutic, prophylactic and/or diagnostic agents. Such recombinant products may include protein products, preferably animal and plant derived products, that are not reporter or marker gene products, including mammalian gene products such as human or human-like protein products.